In recent years, biosensors have been used in the field of medicine and others. Targets to be measured by biosensors are various chemical substances, examples of which include low molecular weight and high molecular weight molecules. In accordance with the target to be measured, the development of a biosensor having various functions has been advanced.
Hitherto, biosensors have been known which can easily attain quantitative determination without diluting or stirring a specific component (substrate) contained in living body sample, or food. Suggested is, for example, a biosensor obtained by: forming an electrode system having at least a working electrode and a counter electrode on an insulating base plate; laying, onto this electrode system, an enzyme reaction layer containing a redox enzyme and an electron receptor each immobilized with an immobilizing agent such as a hydrophilic polymer; next laying a filtrating layer (blood corpuscle removing layer) onto this enzyme reaction layer; and further covering this filtrating layer, from the above, with a cover to integrate these members into a unit.
By such biosensor, the concentration of substrate in a sample is quantitatively determined in the following manner: First, a sample solution, such as blood, is dropped onto the filtrating layer, and the resultant filtrate penetrates into the enzyme reaction layer. In this way, the redox enzyme and the electron receptor are dissolved in the sample solution so that an enzyme reaction advances between the substrate and the enzyme. By this enzyme reaction, the substrate is oxidized and simultaneously the electron receptor is reduced. After the enzyme reaction ends, the reduced electron receptor is electrochemically oxidized. From the oxidation current value obtained at this time, the concentration of the substrate in the sample solution can be calculated.
As a method for measuring neutral fat with biosensor, known is, for example, a method of determining neutral fat in sample quantitatively as follows: the neutral fat contained in sample solution is first decomposed into, for example, free fatty acid and glycerol with lipoprotein lipase (LPL). As shown in formulae (1) and (2) described below, glycerol generated therein can be quantitatively determined, using glycerol kinase (GK), and glycerol-3-phosphoric acid oxidase (GPO), or glycerol-3-phosphoric acid dehydrogenase (GPDH). In other words, the glycerol can be quantitatively determined by measuring decrease in the oxidized-form electron receptor, increase in the reduced-form electron receptor, or the quantity of dihydroxyacetone phosphoric acid, as shown in the following formulae. In particular, by measuring the quantity of the increase in the reduced-form electron receptor electrochemically, the glycerol can be quantitatively determined.

However, each of three enzymes, i.e., lipoprotein lipase (LPL), glycerol kinase (GK), and glycerol-3-phosphoric acid oxidase (GPO), which are used in the above-mentioned neutral-fat-measurement is expensive.
In order to solve this problem, a biosensor, in which, as enzymes used in a decomposition reaction of neutral fat, two of neutral fat decomposing enzyme and glycerol dehydrogenase (GLDH) are used to decrease enzyme costs, is disclosed (Patent Literature 1). However, the biosensor of Patent Literature 1 is not sufficient about the measurement time, and in precision. Thus, it is desired to make the precision higher, and make the measurement more rapidly.
In the meantime, as a method using a single enzyme without effect of dissolved oxygen, known is a method as shown in a formula (3) described below, wherein NAD+ dependent glycerol dehydrogenase (NAD-GLDH) is used.

However, this reaction requires the addition of NAD+, which is expensive.
As a method for determining glycerol quantitatively, easily and inexpensively, known is a method using polyol dehydrogenase into which pyrroloquninoline quinone is incorporated as a prosthetic group (PQQ-PDH).
This method is performed in accordance with a reaction of a formula (4) described below; thus, the method has, for example, advantages that the determination is not affected by any dissolved oxygen, the reaction is simple, the use of plural enzymes is unnecessary, and the addition of expensive NAD+ is not required.

Considering the above matters, in order to supply a biosensor capable of measuring neutral fat with high precision in short time, reports have been hitherto made about biosensors in each of which a neutral fat decomposing enzyme and glycerol dehydrogenase are located indifferent layers, respectively (Patent Literatures 2 and 3). The biosensor of Patent Literature 2 has a structure having, on an electrode, a polymer layer containing GLDH and a hydrophilic polymer, and a filter layer containing a neutral fat decomposing enzyme carried on a filter paper, these two reaction layers being laminated in turn. The biosensor of Patent Literature 3 is characterized by having a structure having, on an electrode, a polymer layer containing GLDH and a hydrophilic polymer, and a nonwoven cloth layer containing a neutral fat decomposing enzyme carried on a nonwoven cloth, these two reaction layers being laminated in turn.